Total RNA Purification Maxi Kit - Geneflow Home
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1. e Centrifuge at 300 x g 800 to 1 000 RPM for 5 minutes and decant supernatant A few uL of media may be left behind with the pellet in order to ensure that the pellet is not dislodged f Add 8 mL of Lysis Solution directly to pelleted leukocytes g Lyse cells by gentle vortexing until homogeneity is reached h Add 4 5 mL of 95 100 ethanol provided by the user to the mixture and mix by vortexing for 10 seconds Note For input amounts greater than 3 x 10 leukocytes it is recommended that the lysate is passed through a 25 gauge needle attached to a syringe 5 10 times at this point in order to shear the genomic DNA prior to loading onto the column 1E Lysate Preparation from Bacteria Notes Prior to Use 1E Prepare the appropriate lysozyme containing TE Buffer as indicated in Table 2 This solution should be prepared with sterile RNAse free TE Buffer and kept on ice until needed These reagents are to be provided by the user It is recommended that no more than 2 5 x 10 bacterial cells 25 mL E coli be used in this procedure Bacterial growth can be measured using a spectrophotometer As a general rule an E coli culture containing 1 x 10 cells mL has an ODs of 1 0 For RNA isolation bacteria should be harvested in log phase growth Bacterial pellets can be stored at 70 C for later use or used directly in this procedure Frozen bacterial pellets should not be thawed prior to beginning the protocol Add th2. Yeast can be stored at 70 C for later use or used directly in this procedure Frozen yeast pellets should not be thawed prior to beginning the protocol Add the Lyticase containing Resuspension Buffer directly to the frozen yeast pellet Step 1Fc 1F Cell Lysate Preparation oS Pellet yeast by centrifuging at 3 000 x g 3 000 RPM for 5 minutes Decant the supernatant and carefully remove any remaining media by aspiration Resuspend the yeast thoroughly in 2 mL of Lyticase containing Resuspension Buffer by vortexing Incubate at 37 C for 10 minutes Add 6 mL of Lysis Solution and vortex vigorously for at least 10 seconds Add 4 mL of 95 100 ethanol provided by the user to the lysate Mix by vortexing for 10 seconds Proceed to Step 2 1G Lysate Preparation from Fungi Notes Prior to Use Fresh or frozen fungi may be used for this procedure Fungal tissues should be flash frozen in liquid nitrogen and transferred immediately to a 70 C freezer for long term storage Fungi may be stored at 70 C for several months Do not allow frozen tissues to thaw prior to grinding with the mortar and pestle in order to ensure that the integrity of the RNA is not compromised lt is recommended that no more than 1 g of fungi be used for this procedure in order to prevent clogging of the column 10 1G Cell Lysate Preparation from Fungi Determine the amount of fungi by weighing It is recommended that no more than 1 g of fungi be
3. gt 50 mg of connective tissues However it is recommended that the maximum tissue input does not exceed 250 mg Cell Lysate Preparation ze Excise the tissue sample from the animal Determine the amount of tissue by weighing Please refer to Table 1 for the recommended maximum input amounts of different tissues For tissues not included in the table we recommend starting with an input of no more than 100 mg Transfer the tissue into a mortar that contains an appropriate amount of liquid nitrogen to cover the sample Grind the tissue thoroughly using a pestle Note The use of liquid nitrogen is recommended However if homogenization without flash freezing is preferred proceed to Step e Allow the liquid nitrogen to evaporate without allowing the tissue to thaw Add 4 mL of Lysis Solution to the tissue sample and continue to grind until the sample has been homogenized Note Maximum homogenization may be achieved by passing the lysate 5 10 times through a 25 gauge needle attached to a syringe Using a pipette transfer the lysate into an RNase free 50 mL tube not provided Add 8 mL of RNase Free Water not provided to the lysate Vortex to mix Add 250 uL of reconstituted Proteinase K to the lysate and incubate at 55 C for 15 minutes Vortex the tubes occasionally during incubation Spin the lysate for 10 minutes at 3 000 x g 3 000 RPM to pellet any cell debris Transfer the supernatant to another RNase free 50 mL centrifu
4. at least 1 year in their unopened containers Precautions and Disclaimers This kit is designed for research purposes only It is not intended for human or diagnostic use Ensure that a suitable lab coat disposable gloves and protective goggles are worn when working with chemicals For more information please consult the appropriate Material Safety Data Sheets MSDSs These are available as convenient PDF files online at www norgenbiotek com Blood of all human and animal subjects is considered potentially infectious All necessary precautions recommended by the appropriate authorities in the country of use should be taken when working with whole blood Customer Supplied Reagents and Equipment You must have the following in order to use the Total RNA Purification Maxi Kit For All Protocols e Centrifuge with a swinging bucket rotor capable of 3000 x g e 95 100 ethanol e B mercaptoethanol optional For Animal Cell Protocol e PBS RNase free For Animal Tissue Protocol Liquid nitrogen Mortar and pestle 70 ethanol For Leukocytes RBC Lysis Buffer Norgen Cat 21201 For Bacterial Protocol Lysozyme containing TE Buffer o For Gram negative bacteria 1 mg mL lysozyme in TE Buffer o For Gram positive bacteria 3 mg mL lysozyme in TE Buffer For Yeast Protocol Resuspension Buffer with Lyticase 50 mM Tris pH 7 5 o 10mMEDTA o 1M Sorbital o 1 unit uL Lyticase O For Fungi Protocol Liquid nitrogen Mort
5. be used in order to prevent clogging of the column e It is important to work quickly during this procedure 11 Cell Lysate Preparation from Viral Suspension a Transfer up to 2 mL of viral suspension to an RNase free 50 mL centrifuge tube not provided b Add 6mL of Lysis Solution Lyse viral cells by vortexing for 15 seconds Ensure that mixture becomes transparent before proceeding to the next step c Add 4 mL of 95 100 ethanol provided by the user to the lysate Mix by vortexing for 10 seconds Proceed to Step 2 Section 2 Total RNA Purification from All Types of Lysate Notes e The remaining steps of the procedure for the purification of total RNA are the same from this point forward for all the different types of lysate e Preheat an appropriate amount of Elution Solution to 70 C prior to isolation 2 Binding RNA to Column a Apply all of the lysate with the ethanol from Step 1 onto the RNA maxi spin column assembly Place the cap loosely onto the column assembly so as not to impede liquid flow Centrifuge at 3 000 x g 3 000 RPM for 5 minutes Note Ensure the entire lysate volume has passed through into the collection tube by inspecting the column If the entire lysate volume has not passed spin for an additional 5 minutes b Discard the flowthrough Reassemble the spin column with its collection tube Optional Step Norgen s Total RNA Purification Maxi Kit isolates total RNA with minimal amounts of ge
6. check literature to determine the expected RNA content of your starting material Ensure that the cell monolayer is washed with the appropriate amount of PBS in order to remove residual media from cells Ensure that the appropriate amount of lyticase is added when making the Resuspension Buffer Ensure that all media is removed prior to the addition of the lysis solution through aspiration Ensure that the appropriate amount of lysis buffer was used for the amount of cells or tissue Refer to specifications to determine if amount of starting material falls within kit specifications The lysate may be passed through a 25 gauge needle attached to a syringe 5 10 times in order to shear the genomic DNA prior to loading onto the column Ensure that the appropriate amount of Lysis Solution was used for the amount of cells or tissue 16 Possible Cause Solution and Explanation Clogged Column RNA is Degraded RNA does not perform well in downstream applications Centrifuge temperature too low RNA Maxi Column Assembly was tightly capped RNase contamination Procedure not performed quickly enough Improper storage of the purified RNA Frozen tissues or cell pellets were allowed to thaw prior to RNA isolation Starting material may have a high RNase content Lysozyme or lyticase used may not be RNAse free RNA was not washed 2 times with the provided Wash Solution Ethanol carryover Ensure tha
7. in a centrifuge with a swinging bucket Various speeds are required for different steps so please check your centrifuge specifications to ensure that it is capable of the proper speeds All centrifugation steps are performed at room temperature The correct rom can be calculated using the formula RPM RCF 1 118 x 105 r where RCF required gravitational acceleration relative centrifugal force in units of g r radius of the rotor in cm and RPM the number of revolutions per minute required to achieve the necessary g force Section 1 Preparation of Lysate From Various Cell Types Notes Prior to Use e The steps for preparing the lysate are different depending on the starting material Step 1 However the subsequent steps are the same in all cases Steps 2 6 e Please ensure that the correct procedure for preparing the lysate from your starting material is followed e All centrifugation steps are carried out in a centrifuge with a swinging bucket at 3 000 x g 3 000 RPM except where noted All centrifugation steps are performed at room temperature e Ensure that all solutions are at room temperature prior to use e Prepare a working concentration of the Wash Solution by adding 50 mL of 95 ethanol provided by the user to each of the four supplied bottles containing the concentrated Wash Solution This will give a final volume of 72 mL per bottle The label on the bottles has a box that may be checked to indicate th
8. procedure 1H Cell Lysate Preparation from Plant a Transfer lt 1 g of plant tissue or 1 x 10 plant cells into a mortar that contains an appropriate amount of liquid nitrogen to cover the sample Grind the sample into a fine powder using a pestle in liquid nitrogen Note If stored frozen samples are used do not allow the samples to thaw before transferring to the liquid nitrogen Allow the liquid nitrogen to evaporate without allowing the tissue to thaw Add 8 mL of Lysis Solution to the tissue sample and continue to grind until the sample has been homogenized Using a pipette transfer the lysate into an RNase free 50 mL centrifuge tube not provided Spin the lysate for 5 minutes to pellet any cell debris Transfer the supernatant to another RNase free 50 mL centrifuge tube Note the volume of the supernatant lysate Add an equal volume of 70 ethanol provided by the user that is equivalent to the lysate volume collected 1 mL of 70 ethanol is added to every 1 mL of lysate Vortex to mix Proceed to Step 2 1 11 Lysate Preparation from Viruses Notes Prior to Use e For the isolation of integrated viral RNA follow Section 1A if the starting material is cell culture follow Section 1B if the starting material is tissue follow Section 1C if the starting material is blood e For the isolation of RNA from free viral particles follow the procedure below e t is recommended that no more than 2 mL of viral suspension
9. used for the protocol Transfer the fungus into a mortar that contains an appropriate amount of liquid nitrogen to cover the sample Grind the fungus thoroughly using a pestle Note At this stage the ground fungus may be stored at 70 C such that the RNA purification can be performed at a later time Allow the liquid nitrogen to evaporate without allowing the tissue to thaw Add 8 mL of Lysis Solution to the tissue sample and continue to grind until the sample has been homogenized Using a pipette transfer the lysate into an RNase free 50 mL centrifuge tube not provided Spin the lysate for 5 minutes to pellet any cell debris Transfer the supernatant to another RNase free 50 mL centrifuge tube Note the volume of the supernatant lysate Add an equal volume of 70 ethanol provided by the user that is equivalent to the lysate volume collected 1 mL of ethanol is added to every 1 mL of lysate Vortex to mix Proceed to Step 2 1H Lysate Preparation from Plant Notes Prior to Use The maximum recommended input of plant tissue is 1 g or 1 x 10 plant cells Both fresh and frozen plant samples can be used for this protocol Samples should be flash frozen in liquid nitrogen and transferred immediately to a 70 C freezer for long term storage Do not allow frozen tissues to thaw prior to grinding with the mortar and pestle in order to ensure that the integrity of the RNA is not compromised lt is important to work quickly during this
10. Fax 905 227 1061 BIOTEK wi CORPORATION Email techsupport norgenbiotek com gt k 3430 Schmon Parkway lt Thorold ON Canada L2V 4Y6 gt Phone 866 667 4362 e 905 227 8848 Total RNA Purification Maxi Kit Product Insert Product 26800 Norgen s Total RNA Purification Maxi Kit provides a rapid method for the isolation and purification of total RNA from cultured animal cells tissue samples blood bacteria yeast fungi plants and viruses The kit purifies all sizes of RNA from large mRNA and ribosomal RNA down to microRNA miRNA and small interfering RNA siRNA The RNA is preferentially purified from other cellular components such as proteins without the use of phenol or chloroform The purified RNA is of the highest integrity and can be used in a number of downstream applications including real time PCR reverse transcription PCR Northern blotting RNase protection and primer extension and expression array assays Norgen s Purification Technology Purification is based on spin column chromatography using Norgen s proprietary resin as the separation matrix The RNA is preferentially purified from other cellular components such as proteins without the use of phenol or chloroform The process involves first lysing the cells or tissue of interest with the provided Lysis Solution please see the flow chart on page 4 Ethanol is then added to the lysate and the solution is loaded onto a maxi spin column Norgen s resin bind
11. ar and pestle 70 ethanol For Plant Protocol Liquid nitrogen Mortar and pestle 70 ethanol Working with RNA RNases are very stable and robust enzymes that degrade RNA Autoclaving solutions and glassware is not always sufficient to actively remove these enzymes The first step when preparing to work with RNA is to create an RNase free environment The following precautions are recommended as your best defense against these enzymes The RNA area should be located away from microbiological work stations Clean disposable gloves should be worn at all times when handling reagents samples pipettes disposable tubes etc It is recommended that gloves are changed frequently to avoid contamination There should be designated solutions tips tubes lab coats pipettes etc for RNA only All RNA solutions should be prepared using at least 0 05 DEPC treated autoclaved water or molecular biology grade nuclease free water Clean all surfaces with commercially available RNase decontamination solutions When working with purified RNA samples ensure that they remain on ice during downstream applications Flowchart Procedure for Purifying Total RNA using Norgen s Total RNA Purification Maxi Kit Lyse cells or tissue using Lysis Solution i Add Ethanol Bind to column lt Wash three times with Wash Solution Elute RNA with Elution Solution SPIN 3 Purified Total RNA Procedures All centrifugation steps are carried out
12. at the ethanol has been added e Optional The use of B mercaptoethanol in lysis is highly recommended for most animal tissues particularly those known to have a high RNAse content ex pancreas as well as for most plant tissues and nasal and throat swabs It is also recommended for users who wish to isolate RNA for sensitive downstream applications Add 10 uL of B mercaptoethanol provided by the user to each 1 mL of Lysis Solution required 8 mercaptoethanol is toxic and should be dispensed in a fume hood Alternatively the lysis solution can be used as provided e For the isolation of integrated viral RNA follow Section 1A if the starting material is cell culture follow Section 1B if the starting material is tissue follow Section 1C if the starting material is blood For the isolation of RNA from free viral particles follow Section 1l e Pre warm an appropriate amount of Elution Solution to 70 C prior to isolation e It is important to work quickly during this procedure 1A Lysate Preparation from Cultured Animal Cells Notes Prior to Use The maximum recommended input of cells is 5 x 10 A hemocytometer can be used in conjunction with a microscope to count the number of cells As a general guideline a confluent 15 cm plate of HeLa cells will contain 2 x1 0 cells Cell pellets can be stored at 70 C for later use or used directly in the procedure Determine the number of cells present before freezing Frozen pellets should b
13. e Lysozyme containing TE Buffer directly to the frozen bacterial pellet Step 1Ec Cell Lysate Preparation from Bacteria ao Pellet bacteria by centrifuging at 3 000 x g 3 000 RPM for 5 minutes Decant supernatant and carefully remove any remaining media by aspiration Resuspend the bacteria thoroughly in 2 mL of the appropriate lysozyme containing TE buffer see Table 2 by vortexing Incubate at room temperature for the time indicated in Table 1 Add 6 mL of Lysis Solution and vortex vigorously for at least 10 seconds Add 4 mL of 95 100 ethanol provided by the user to the lysate Mix by vortexing for 10 seconds Proceed to Step 2 Table 2 Incubation Time for Different Bacterial Strains Lysozyme Concentration i i Bacteria Type y A e Incubation Time EE on 1F Lysate Preparation from Yeast Notes Prior to Use Prepare the appropriate amount of Lyticase containing Resuspension Buffer considering that 2 mL of buffer is required for each preparation The Resuspension Buffer should have the following composition 50 mM Tris pH 7 5 10 mM EDTA 1M Sorbital 0 1 B mercaptoethanol and 1 unit uL Lyticase This solution should be prepared with sterile RNAse free reagents and kept on ice until needed These reagents are to be provided by the user It is recommended that no more than 2 x 10 yeast cells or 20 mL of culture be used for this procedure For RNA isolation yeast should be harvested in log phase growth
14. e stored for no longer than 2 weeks to ensure that the integrity of the RNA is not compromised Frozen cell pellets should not be thawed prior to beginning the protocol Add the Lysis Solution directly to the frozen cell pellet Step 1A ii c 1A i Cell Lysate Preparation from Cells Growing in a Monolayer 1A ii Aspirate media and wash cell monolayer with an appropriate amount of PBS Aspirate PBS Add a total of 8 mL of Lysis Solution directly to culture plate s A minimum volume of 3 mL of Lysis Solution should be added to each 15 cm plate Lyse cells by gently tapping culture dish and swirling buffer around plate surface for five minutes Transfer lysate to a 50 mL centrifuge tube Add 4 5 mL of 95 100 ethanol provided by the user to the 8 mL lysate Mix by vortexing for 10 seconds Proceed to Step 2 Note For input amounts greater than 3 x 10 cells it is recommended that the lysate is passed through a 25 gauge needle attached to a syringe 5 10 times at this point in order to shear the genomic DNA prior to loading onto the column Cell Lysate Preparation from Cells Growing in Suspension and Lifted Cells Transfer cell suspension to an RNase free tube not provided and centrifuge at no more than 300 x g 800 to 1 000 RPM for 10 minutes to pellet cells Carefully decant the supernatant A few uL of media may be left behind with the pellet in order to ensure that the pellet is not dislodged Add 8 mL of Lysis Solu
15. e with downstream applications and thus must be washed from the column Ensure that the dry spin under the Column Wash procedure is performed in order to remove traces of ethanol prior to elution Ethanol is Known to interfere with many downstream applications 17 Possible Cause Solution and Explanation Perform RNAse free DNasel digestion on the RNA sample after elution to remove genomic DNA contamination It is recommended that Norgen s RNase Free DNase Kit Product 25710 be used for this step Genomic Large amounts of DNA starting material contamination used Technical Support Contact our Technical Support Team between the hours of 8 30 and 5 30 Eastern Standard Time at 905 227 8848 or Toll Free at 1 866 667 4362 Technical support can also be obtained from our website www norgenbiotek com or through email at techsupport norgenbiotek com 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 Phone 905 227 8848 Fax 905 227 1061 Toll Free in North America 1 866 667 4362 2009 Norgen Biotek Corp P1l126800 3 18
16. ge tube Add 6 mL of 95 ethanol provided by the user to the lysate Vortex to mix 15 Troubleshooting Guide Possible Cause Solution and Explanation Incomplete lysis of cells or tissue Poor RNA Recovery Clogged Column Column has become clogged An alternative elution solution was used Ethanol was not added to the lysate Ethanol was not added to the Wash Solution Low RNA content in cells or tissues used Cell Culture Cell monolayer was not washed with PBS Yeast Lyticase was not added to the Resuspension Buffer Bacteria and Yeast All traces of media not removed Insufficient solubilization of cells or tissues Maximum number of cells or amount of tissue exceeds kit specifications High amounts of genomic DNA present in sample Do not exceed the recommended amounts of starting materials The amount of starting material may need to be decreased if the column shows clogging below the recommended levels See also Clogged Column below It is recommended that the Elution Solution supplied with this kit be used for maximum RNA recovery Ensure that the appropriate amount of ethanol is added to the lysate before binding to the column Ensure that 50 mL of 95 ethanol is added to each of the supplied Wash Solution bottle prior to use Different tissues and cells have different RNA contents and thus the expected yield of RNA will vary greatly from these different sources Please
17. h Solution to the column and centrifuge at 3 000 x g 3 000 RPM for 5 minutes Discard the flowthrough Reassemble the spin column with its collection tube Apply 1 mL of the RNase free DNase solution prepared in Step 1 to the column and centrifuge at 3 000 x g 3 000 RPM for 5 minutes Note Ensure that the entire DNase solution passes through the column If needed spin at 3 000 x g 3 000 RPM for an additional two minutes After the centrifugation in Step 4 pipette the flowthrough that is present in the collection tube back onto the top of the column Note Ensure Step 5 is performed in order to ensure maximum DNase activity and to obtain maximum yields of RNA in particular for small RNA species Incubate the column assembly at 25 30 C for 15 minutes Without any further centrifugation apply 7 5 mL of Wash Solution to the column and centrifuge at 3 000 x g 3 000 RPM for 5 minutes Discard the flowthrough Reassemble the spin column with its collection tube Apply 15 mL of Wash Solution to the column and centrifuge for 5 minute Discard the flowthrough and reassemble the spin column with its collection tube Spin the column for 5 minutes in order to thoroughly dry the resin Discard the collection tube Proceed to Step 4 RNA Elution Appendix B Lysate Preparation from Animal Tissues with the use of Proteinase K Customer Supplied Reagent RNase Free Proteinase K RNase Free Water Notes Prior to Use Ensu
18. nomic DNA contamination However an optional On Column DNA Removal Protocol is provided in Appendix A for maximum removal of residual DNA that may affect sensitive downstream applications It is recommended that Norgen s RNase Free DNase Kit Product 25710 be used for this step This step should be performed at this point in the protocol 12 3 Column Wash a Apply 15 mL of Wash Solution to the column assembly Place the cap loosely onto the column assembly so as not to impede liquid flow Centrifuge at 3 000 x g 3 000 RPM for 5 minutes Note Ensure the entire wash solution has passed through into the collection tube by inspecting the column If the entire wash volume has not passed spin for an additional minute b Discard the flowthrough and reassemble the spin column with its collection tube c Repeat steps 3a and 3b to wash column a second time d Spin the column for 5 minutes in order to thoroughly dry the resin Discard the collection tube 4 RNA Elution a Ensure that the Elution Solution is heated to 70 C b Place the column into a fresh 50 mL Elution Tube provided with the kit c Add 2 mL of warm Elution Solution to the column Place the cap loosely onto the column assembly so as not to impede liquid flow d Centrifuge for 5 minutes at 3 000 x g 3 000 RPM Note the volume eluted from the column If the entire 2 mL has not been eluted spin the column at 3 000 x g 3 000 RPM for an additional 5 mi
19. nutes Note For maximum RNA recovery it is recommended that a second elution be performed into a separate 50 mL centrifuge tube Repeat Steps 4c and 4d 5 Storage of RNA The purified RNA sample may be stored at 20 C for a few days It is recommended that samples be placed at 70 C for long term storage Appendix A Protocol for Optional On Column DNA Removal Norgen s Total RNA Purification Maxi Kit isolates total RNA with minimal amounts of genomic DNA contamination However an optional protocol is provided below for maximum removal of residual DNA that may affect sensitive downstream applications It is recommended that Norgen s RNase free DNase Kit Product 25710 be used for this step Each RNase free DNase Kit will be sufficient to perform four on column DNA removals 1 For every on column reaction to be performed prepare a mix of 150 uL of DNase I and 1 mL of Enzyme Incubation Buffer using Norgen s RNase Free DNase Kit Product 25710 Mix gently by inverting the tube a few times DO NOT VORTEX Note If using an alternative DNase I prepare a working stock of 0 25 Kunitz unit uL RNase free DNase solution according to the manufacturer s instructions A 1 mL aliquot is required for each column to be treated 13 Perform the appropriate Total RNA Isolation Procedure for your starting material up to and including Binding to Column Steps 1 and 2 of all protocols Apply 7 5 mL of Was
20. protocol is 10 mL of blood Please note that the RBC Lysis Buffer Cat 21201 is to be provided by the user e The maximum recommended input of leukocytes is 5 x 10 As a general guideline blood from a healthy individual contains about 5 x 10 leukocyte per mL e If the leukocytes were purified by other methods ensure that either the cells are used fresh proceed to Step 1D f or flash frozen by liquid nitrogen before use e Frozen pellets should be stored for no longer than 2 weeks to ensure that the integrity of the RNA is not compromised e Frozen cell pellets should not be thawed prior to beginning the protocol Add the Lysis Solution directly to the frozen cell pellet Step 1D f 1D Lysate Preparation from Leukocytes a Add 5 volumes of RBC Lysis Buffer provided by the user to blood samples collected with EDTA i e Add 5 mL of RBC Lysis Buffer to 1 mL of blood Up to 10 mL of blood from a healthy individual may be used as the input b Incubate at room temperature for 3 to 5 minutes with brief vortexing during the incubation to mix Note Ensure that the solution changes from a milky opaque pink to clear red before proceeding to the next step c Centrifuge at 300 x g 800 to 1 000 RPM for 5 minutes and decant the supernatant d Add 2 additional volumes of RBC Lysis Buffer to pelleted white blood cells and mix by gentle vortexing for 10 seconds i e Add 2 mL of RBC Lysis Buffer to every 1 mL of input blood volume
21. re that all solutions are at room temperature prior to use All enzymes provided should remain at the storage temperature indicated on each vial until use Reconstitute each of the Proteinase K vials in an appropriate amount of molecular biology grade water or 10 mM Tris HCl pH 7 5 RNase Free to give a 20 mg mL final concentration Aliquot into small fractions and store the unused portions at 20 C until needed Add 10 uL of B mercaptoethanol provided by the user to each 1 mL of Lysis Solution required B mercaptoethanol is toxic and should be dispensed in a fume hood RNA in animal tissues is not protected after harvesting until it is disrupted and homogenized Thus it is important that the procedure is carried out as quickly as possible particularly the Cell Lysate Preparation step Fresh or frozen tissues may be used for the procedure Tissues should be flash frozen in liquid nitrogen and transferred immediately to a 70 C freezer for long term storage Tissues may be stored at 70 C for several months When isolating total RNA from frozen tissues ensure that the tissue does not thaw during weighing or prior to grinding with the mortar and pestle 14 Tissues stored in RNA stabilization reagents such as RNAlater are compatible with this isolation procedure Prior to isolation carefully remove the tissue from the storage reagent using forceps and dry any excessive liquid This protocol is particularly suitable for isolating RNA from
22. s RNA in a manner that depends on ionic concentrations Thus only the RNA will bind to the column while the contaminating proteins will be removed in the flowthrough or retained on the top of the resin The bound RNA is then washed with the provided Wash Solution in order to remove any remaining impurities and the purified total RNA is eluted with the Elution Solution The purified RNA is of the highest integrity and can be used in a number of downstream applications Specifications Kit Specifications Maximum Column Loading Volume All sizes including small RNA Size of RNA Purified lt 200 nt Maximum Amount of Starting Material Animal Cells 5 x 10 cells Animal Tissues 50 250 mg Blood 2 10 mL Bacteria 2 5 x 10 cells Yeast 2 x 10 cells Fungi Plant Tissues g g j Time to Complete 4 Purifications Average Yields HeLa Cells 5 x 10 cells 750 ug E coli 2 5 x 10 cells 1 5 mg for isolating total RNA from purified leukocytes Advantages e Fast and easy processing using rapid spin column format e Isolate total RNA from large rRNA down to microRNA miRNA e No phenol or chloroform extractions e Isolate high quality total RNA from a variety of sources Kit Components RNA Maxi Spin Columns with collection tubes Elution tubes 50 mL a Storage Conditions and Product Stability All solutions should be kept tightly sealed and stored at room temperature These reagents should remain stable for
23. sue being used Please refer to Table 1 below as a guideline for maximum tissue input amounts If your tissue of interest is not included in the table below we recommend starting with an input of no more than 100 mg To isolate RNA from larger amounts of tissue particularly from those with high connective tissue content such as muscle and heart please follow the specialized protocol with the use of Proteinase K in Appendix B Table 1 Recommended Maximum Input Amounts of Different Tissues 1B Cell Lysate Preparation from Animal Tissues Excise the tissue sample from the animal Determine the amount of tissue by weighing Please refer to Table 1 for the recommended maximum input amounts of different tissues For tissues not included in the table we recommend starting with an input of no more than 100 mg Transfer the tissue into a mortar that contains an appropriate amount of liquid nitrogen to cover the sample Grind the tissue thoroughly using a pestle Allow the liquid nitrogen to evaporate without allowing the tissue to thaw Add 8 mL of Lysis Solution to the tissue sample and continue to grind until the sample has been homogenized Homogenize by passing the lysate 5 10 times through a 25 gauge needle attached to a syringe Using a pipette transfer the lysate into an RNase free 50 mL centrifuge tube not provided g Spin the lysate for 10 minutes at 3 000 x g 3 000 RPM to pellet any cell debris Transfer the supernatant to ano
24. t the centrifuge remains at room temperature throughout the procedure Temperatures below 15 C may cause precipitates to form that can cause the columns to clog Ensure that the cap is loosely placed onto the column assembly so as not to impede liquid flow RNases may be introduced during the use of the kit Ensure proper procedures are followed when working with RNA Please refer to Working with RNA at the beginning of this user guide In order to maintain the integrity of the RNA it is important that the procedure be performed quickly This is especially important for the Cell Lysate Preparation Step in the Animal Tissue protocol since the RNA in animal tissues is not protected after harvesting until it is disrupted and homogenized For short term storage RNA samples may be stored at 20 C for a few days It is recommended that samples be stored at 70 C for longer term storage Do not allow frozen tissues to thaw prior to grinding with the mortar and pestle in order to ensure that the integrity of the RNA is not compromised For starting materials with high RNAase content it is recommended that B mercaptoethanol be added to the Lysis Solution Ensure that the lysozyme and lyticase being used with this kit is RNase free in order to prevent possible problems with RNA degradation Traces of salt from the binding step may remain in the sample if the column is not washed 2 times with Wash Solution Salt may interfer
25. ther RNase free 50 mL centrifuge tube Note the volume of the supernatant lysate h Add an equal volume of 70 ethanol provided by the user to the lysate volume collected 1 mL of 70 ethanol is added to every 1 mL of clarified lysate Vortex to mix Proceed to Step 2 1C Lysate Preparation from Blood Notes Prior to Use e Blood of all human and animal subjects is considered potentially infectious All necessary precautions recommended by the appropriate authorities in the country of use should be taken when working with whole blood e It is recommended that no more than 2 mL of blood be used in order to prevent clogging of the column If you wish to isolate RNA from greater than 2 mL please follow the protocol in Section 1D below e We recommend the use of this kit to isolate RNA from non coagulating fresh blood using EDTA as the anti coagulant e It is important to work quickly during this procedure 1C Cell Lysate Preparation from Blood a Transfer up to 2 mL of non coagulating blood to an RNase free 50 mL centrifuge tube not provided b Add 6 mL of Lysis Solution to the blood Lyse cells by vortexing for 15 seconds Ensure that mixture becomes transparent before proceeding to the next step c Add 4 mL of 95 100 ethanol provided by the user to the lysate Mix by vortexing for 10 seconds Proceed to Step 2 1D Lysate Preparation from Leukocytes Notes Prior to Use e The maximum recommended input for the following
26. tion to the pellet Lyse cells by vortexing for 15 seconds Ensure that the entire pellet is completely dissolved before proceeding to the next step Add 4 5 mL of 95 100 ethanol provided by the user to the lysate Mix by vortexing for 10 seconds Proceed to Step 2 Note For input amounts greater than 3 x 10 cells it is recommended that the lysate is passed through a 25 gauge needle attached to a syringe 5 10 times at this point in order to shear the genomic DNA prior to loading onto the column 1B Lysate Preparation from Animal Tissues Notes Prior to Use RNA in animal tissues is not protected after harvesting until it is disrupted and homogenized Thus it is important that the procedure is carried out as quickly as possible particularly the Cell Lysate Preparation step Fresh or frozen tissues may be used for the procedure Tissues should be flash frozen in liquid nitrogen and transferred immediately to a 70 C freezer for long term storage Tissues may be stored at 70 C for several months When isolating total RNA from frozen tissues ensure that the tissue does not thaw during weighing or prior to grinding with the mortar and pestle Tissues stored in RNA stabilization reagents such as RNAlater are compatible with this isolation procedure Prior to isolation carefully remove the tissue from the storage reagent using forceps and dry excessive liquid The maximum recommended input of tissue varies depending on the type of tis
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